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Metabolic analysis of shikimic acid producing Escherichia coli

Johansson, Louise LU (2006)
Abstract (Swedish)
Popular Abstract in Swedish

Efterfrågan på shikimsyra, vilken återfinns i biosyntesvägen för bl.a. de aromatiska aminosyrorna tryptofan, fenylalanin och tyrosin, har under de senare åren ökat. Orsaken till detta är mestadels att den är råmatrial till antiinfluensamedicinen Tamiflu?. Den ökade efterfrågan har drivit fram nya tillverkningsmetoder såsom produktion via E.coli- fermentation. Även om man har lyckats att nå en industriellt gångbar process finns det fortfarande problem rörande E.coli:s shikimsyrametabolism som måste lösas för att nå en optimal produktion. I denna avhandling presenteras en stude där syftet var att öka förståelsen av metabolismen i en shikimsyraproducerande typ av E.coli för att i förlängningen... (More)
Popular Abstract in Swedish

Efterfrågan på shikimsyra, vilken återfinns i biosyntesvägen för bl.a. de aromatiska aminosyrorna tryptofan, fenylalanin och tyrosin, har under de senare åren ökat. Orsaken till detta är mestadels att den är råmatrial till antiinfluensamedicinen Tamiflu?. Den ökade efterfrågan har drivit fram nya tillverkningsmetoder såsom produktion via E.coli- fermentation. Även om man har lyckats att nå en industriellt gångbar process finns det fortfarande problem rörande E.coli:s shikimsyrametabolism som måste lösas för att nå en optimal produktion. I denna avhandling presenteras en stude där syftet var att öka förståelsen av metabolismen i en shikimsyraproducerande typ av E.coli för att i förlängningen möjliggöra en optimering av processen. Olika tekniker såsom traditionella fermentationmetoder, RNA-uttrycksanalys (transcriptome analysis), fluxanalys (MFA) och 2-dimensionell (2-D) fluorometri användes. Fermentation (mestadels kemostat) under kolbegränsning och kolrika förhållanden (mestadels fosfatbegränsning) utgjorde basen för studierna. 2-D-fluorescensmätningar användes främst för att studera stabiliten hos E.coli i kemostatmiljö. Bakgrunden till denna delstudie var ett känt problem av instabilitet av plasmidinnehållande E.coli-stammar, vilket shikimsyrastammen var ett exempel på.



Två huvudproblem, vilka orsakar ett minskat shikimsyrautbyte är kända. Det ena rör biproduktbildning av andra metaboliter i shikimsyravägen (d.v.s. DHS, DHQ och quinat) och det andra rör totalutbytet av alla shikimsyravägsprodukter vilket av någon anledning är mindre under vissa förhållanden. Mekanismerna bakom dessa problem är inte kända men ett par förklaringsmodeller för biproduktsbildningen diskuteras. I denna avhandling visas att kolbegränsning i jämförelse med fosfatbegränsning faktiskt har den största potentialen till optimerad shikimsyraproduktion eftersom totalutbytet här är störst. Dock på bekostnad av en större biproduktbildning.



RNA-expressionsanalysen lyckades ge en ökat förståelse för båda ovan beskrivna problem genom att påvisa en uppreglering av vissa gener under kolbegränsning som kunde kopplas till båda dessa fenomen. Fluxanalysen lyckades ytterligare öka förståelsen genom att modellera mekanismen som styr tillflödet till shikimsyravägen.



Eftersom de använda metoderna är globala i sin natur kunde även fenomen som inte direkt var kopplade till shikimsyraproduktionen studeras. Bland annat kunde ?overflow?-metabolism påvisas under fosfatbegränsning, detta blev särskilt tydligt i fluxanalysen. Expressionsanalysen visade också att cellerna under kolbegränsning kände av en viss brist på de aromatiska aminosyrorna vilket inte var fallet under fosfatbegränsning.



Resultaten från 2-D-fluorescensmätningarna visade att shikimsyrastammen var tillräckligt stabil för att ett tillfredställande steady-state skulle uppnås i kemostaterna, men under alla förhållanden kunde adaptionsprocesser observeras. Tillväxt under selektionstryck ökade inte stamstabiliteten nämnvärt. Det visade sig också att denna teknik kunde påvisa mer information om metabolismförändringar kopplade till adaptionsprocesser än vad de traditionella metoderna lyckades med. (Less)
Abstract
Shikimic acid production using recombinant E.coli has been developed during the past 15 years due to the increased demand of this product, primarily as a starting material for the anti-influensa medicine Tamiflu?. A detailed metabolic analysis of a shikimic acid producing E.coli strain was made in the current work in order to increase the understanding of shikimic acid production and identify means of improving the process. The strain used (called W3110.shik1) was modified for shikimate production by deletion of aroL, and by overexpression of aroFFBR (on a plasmid). Physiological characterizations were made using techniques such as metabolite analysis, stoichiometric network analysis, transcriptome analysis, and in situ 2D-fluorometry at... (More)
Shikimic acid production using recombinant E.coli has been developed during the past 15 years due to the increased demand of this product, primarily as a starting material for the anti-influensa medicine Tamiflu?. A detailed metabolic analysis of a shikimic acid producing E.coli strain was made in the current work in order to increase the understanding of shikimic acid production and identify means of improving the process. The strain used (called W3110.shik1) was modified for shikimate production by deletion of aroL, and by overexpression of aroFFBR (on a plasmid). Physiological characterizations were made using techniques such as metabolite analysis, stoichiometric network analysis, transcriptome analysis, and in situ 2D-fluorometry at different modes of cultivation, and compered to a wild-type strain (W3110).



A high yield of shikimic acid requires both a high flux into the pathway and minimum by-product formation. In carbon-limited chemostat cultivation the total flux into the pathway was almost 2 times higher than the one obtained in phosphorus-limited chemostat cultivation (yields on glucose: ~0.2 vs. 0.1 c-mole/c-mole), but at the expense of increased formation of DHS, DHQ and quinic acid. Typical yields carbon- vs. phosphorus-limitation: shikimate: 0.02 vs. 0.06, DHS 0.03-0.12 (increased with increased growth rate) vs. 0.03, DHQ: 0.02 vs. 0.01, quinic acid: 0.02 vs. 0.01. The batch and fed-batch conditions studied showed similar characteristics as their chemostat dittos. A theory to explain the mechanisms of by-product formation, based on intracellular equilibration, is discussed and compared to the hydroaromatic equilibration theory.



Gene expression patterns of the two growth conditions showed differences in agreement with observed changes in total flux and by-product formation. Under carbon-limitation an upregulation of aroF, aroG, talA and talB agreed with the larger total flux into the pathway, and an upregulation of the genes ydiB, ydiN and aroD may explain the increased by-product formation. A slight starvation of the aromatic amino acids (identified by an induction of the trp and tyr-operons) under carbon-limitation, which was relieved under phosphorus limitation, was observed. The relief could possibly be explained by a simultaneous increased expression of aroK and aroA, thus enabling a higher flux downstream of shikimate.



Metabolic flux analysis showed that the flux into the pathway was mostly dependent on the relationship between consumption and production of erythrose-4-phosphate. In addition, a higher flux connected to overflow metabolism was observed in the central metabolism under phosphorus-limitation. These fluxes were generally lower under carbon-limitation.



2D-fluorometry was used to monitor strain stability in chemostat cultivation. It was shown that strain stability was not maintained for more than about 25 generations, even in selective medium. This method provided additional information about metabolic changes coupled to adaptation mechanisms. For example a change in fluorescence probably coupled to pyridoxal-5-phosphate dependent proteins and FAD-related compounds/proteins was observed. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • Professor Sprenger, Georg, Institut für Microbiologie, Stuttgart, Germany
organization
publishing date
type
Thesis
publication status
published
subject
keywords
Kemiteknik och kemisk teknologi, Biokemi, metabolism, Chemical technology and engineering, transcriptome analysis, phosphorus-limitation, carbon-limitation, fed-batch, chemostat, batch, fermentation technology, Escherichia coli, shikimic acid, Biotechnology, Bioteknik, Metabolism, Biochemistry, principal component analysis, 2D-fluorescence spectrophotometry, metabolic flux analysis, carbon-rich
pages
201 pages
publisher
Chemical Engineering, Lund University
defense location
Room K:A, Center for Chemistry and Chemical Engineering, Getingevägen 60, Faculty of Engineering, Lund University
defense date
2006-06-15 10:15
ISBN
91-7422-116-7
language
English
LU publication?
yes
id
5c802dcd-0f74-40a8-ad4f-877af8502761 (old id 546983)
date added to LUP
2007-10-13 11:04:51
date last changed
2016-09-19 08:45:07
@misc{5c802dcd-0f74-40a8-ad4f-877af8502761,
  abstract     = {Shikimic acid production using recombinant E.coli has been developed during the past 15 years due to the increased demand of this product, primarily as a starting material for the anti-influensa medicine Tamiflu?. A detailed metabolic analysis of a shikimic acid producing E.coli strain was made in the current work in order to increase the understanding of shikimic acid production and identify means of improving the process. The strain used (called W3110.shik1) was modified for shikimate production by deletion of aroL, and by overexpression of aroFFBR (on a plasmid). Physiological characterizations were made using techniques such as metabolite analysis, stoichiometric network analysis, transcriptome analysis, and in situ 2D-fluorometry at different modes of cultivation, and compered to a wild-type strain (W3110).<br/><br>
<br/><br>
A high yield of shikimic acid requires both a high flux into the pathway and minimum by-product formation. In carbon-limited chemostat cultivation the total flux into the pathway was almost 2 times higher than the one obtained in phosphorus-limited chemostat cultivation (yields on glucose: ~0.2 vs. 0.1 c-mole/c-mole), but at the expense of increased formation of DHS, DHQ and quinic acid. Typical yields carbon- vs. phosphorus-limitation: shikimate: 0.02 vs. 0.06, DHS 0.03-0.12 (increased with increased growth rate) vs. 0.03, DHQ: 0.02 vs. 0.01, quinic acid: 0.02 vs. 0.01. The batch and fed-batch conditions studied showed similar characteristics as their chemostat dittos. A theory to explain the mechanisms of by-product formation, based on intracellular equilibration, is discussed and compared to the hydroaromatic equilibration theory.<br/><br>
<br/><br>
Gene expression patterns of the two growth conditions showed differences in agreement with observed changes in total flux and by-product formation. Under carbon-limitation an upregulation of aroF, aroG, talA and talB agreed with the larger total flux into the pathway, and an upregulation of the genes ydiB, ydiN and aroD may explain the increased by-product formation. A slight starvation of the aromatic amino acids (identified by an induction of the trp and tyr-operons) under carbon-limitation, which was relieved under phosphorus limitation, was observed. The relief could possibly be explained by a simultaneous increased expression of aroK and aroA, thus enabling a higher flux downstream of shikimate.<br/><br>
<br/><br>
Metabolic flux analysis showed that the flux into the pathway was mostly dependent on the relationship between consumption and production of erythrose-4-phosphate. In addition, a higher flux connected to overflow metabolism was observed in the central metabolism under phosphorus-limitation. These fluxes were generally lower under carbon-limitation.<br/><br>
<br/><br>
2D-fluorometry was used to monitor strain stability in chemostat cultivation. It was shown that strain stability was not maintained for more than about 25 generations, even in selective medium. This method provided additional information about metabolic changes coupled to adaptation mechanisms. For example a change in fluorescence probably coupled to pyridoxal-5-phosphate dependent proteins and FAD-related compounds/proteins was observed.},
  author       = {Johansson, Louise},
  isbn         = {91-7422-116-7},
  keyword      = {Kemiteknik och kemisk teknologi,Biokemi,metabolism,Chemical technology and engineering,transcriptome analysis,phosphorus-limitation,carbon-limitation,fed-batch,chemostat,batch,fermentation technology,Escherichia coli,shikimic acid,Biotechnology,Bioteknik,Metabolism,Biochemistry,principal component analysis,2D-fluorescence spectrophotometry,metabolic flux analysis,carbon-rich},
  language     = {eng},
  pages        = {201},
  publisher    = {ARRAY(0x88b2c78)},
  title        = {Metabolic analysis of shikimic acid producing Escherichia coli},
  year         = {2006},
}